cAMP is a second messenger which regulates responses to many different hormones and neurotransmitters. Elevated levels of cAMP results in the activation of the cAMP-dependent protein kinase (PKA) which produces a variety of distinct physiological responses. One hypothesis to explain how one enzyme can perform many different functions is that the subcellular localization of PKA is tightly controlled in each different cell type. Accordingly, PKA would be targeted or anchored adjacent to its physiological substrate. PKA consists of four subunits, two regulatory and two catalytic. The anchoring of PKA is believed to be accomplished through the interaction of the regulatory subunit (R) of PKA with specific cellular proteins, referred to as A-Kinase Anchoring Proteins (AKAPs). Although characterization of the protein-protein interaction between PKA and anchoring proteins has been extensive, the physiological function of most AKAPs remains unknown. Granulosa cells provide an ideal system for studying the functional role of AKAPs. It is well recognized that FSH and LH sequentially promote granulosa cell differentiation via a mechanism dependent upon the generation of cAMP and the subsequent activation of PKA. However, the mechanism by which PKA regulates this biological process remains elusive. Two AKAPs have recently been identified in primary granulosa cell cultures whose expression is differentially regulated by follicle stimulating hormone (FSH). The expression of one protein, AKAP 80, is stimulated by FSH, while the induction of the other protein, AKAP 140, is blocked by FSH. FSH also causes the induction of one isoform of PKA, regulatory subunit (RIIbeta) and the translocation of another isoform of PKA (RIIalpha). All of this data is consistent with the hypothesis that FSH promotes a redistribution of PKA, possibly to enable the cells to respond to a second hormone. LH, which also causes an increase in cAMP. To test this hypothesis we propose: l) to isolate and characterize both AKAP 80 and 140; 2) to determine the subcellular location of the AKAPs and RIIalpha and RIIbeta; and 3) to determine the functions of AKAPs 80 and 140 and isoforms of PKA in granulosa cells by either blocking the interaction of PKA with anchoring proteins through the use of anchoring inhibitor peptides or blocking the induction of these proteins through the use of antisense oligonucleotides. These studies will provide insights into the physiologic role of specific AKAPs and also new information on the cellular signaling pathway utilized by FSH and LH in granulosa cells.